Optimisation of SARS-CoV-2 culture from clinical samples for clinical trial applications (preprint)

Clinical trials of SARS-CoV-2 therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using RT-qPCR, which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardised for use as clinical trial endpoints. We report optimisation of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-ACE2-TMPRSS2 (VAT) cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22/32, 68.8%), being able to identify viable virus in 83.3% (20/24) of clinical samples with initial Ct values <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero hSLAM cells provides the highest overall detection of Omicron viral replication (9/31, 29%), detecting live virus in 39.1% (9/23) of clinical samples with initial Ct values < 25. This assessment demonstrates that combining RT-qPCR with virological end point analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimising based on dominant circulating strain.

Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene (preprint)

COVID-19 pandemics is caused by the SARS-CoV-2 virus, whose internalization and infection are mediated by the Angiotensin Converting Enzyme 2 (ACE2). The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking Thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2 associated cytokine storm, the transcription factor p63 and ACE2 are stabilized and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the ACE2gene. We previously showed that p63 is degraded upon Thalidomide treatment, and now found that treatment with this drug—or with its analogue Lenalidomide—downregulates ACE2 through p63 degradation. Finally, we found that Thalidomide treatment reduce in vitro infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of Thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19.

Covid19 and Pregnancy: Infected Mothers and Premature Babies? (preprint)

Introduction: The emergency of the SARS-CoV-2 virus spread and its subsequent global pan-demic have raised significant concerns regarding its impact on pregnancy outcomes. This review aims to summarize the emerging data on the risk of preterm delivery in pregnant women infected with SARS-CoV-2. Materials and Methods: A systematic search was conducted from March 2020 to December 2023 using PubMed, following PRISMA guidelines. Studies correlating maternal COVID-19 infection with preterm birth were included. Results: Thirteen studies were analyzed, indicating a higher incidence of preterm birth in SARS-CoV-2 positive pregnant women compared to controls. The average incidence rate of pre-term birth in infected patients was 18.5%, with a median of 12.75%, while non infected women showed an average incidence of preterm birth of 10% with a median of 8.2%. Discussion: Studies suggest an association between SARS-CoV-2 infection during pregnancy and increased risk of preterm birth and cesarean section. Severity of symptoms and underlying comorbidities further elevate this risk. Notably, infections during the third trimester pose the highest risk of preterm birth. Conclusion: Preventing SARS-CoV-2 infection during pregnancy is crucial to mitigate adverse obstetric outcomes. Close monitoring and tailored interventions for infected pregnant women, particularly those in later trimesters and with comorbidities, are imperative to reduce the risk of preterm birth and improve maternal-fetal outcomes.

COVID-19 Therapeutics Use by Social Deprivation in England, July 2020–April 2023 (preprint)

Coronavirus disease-19 (COVID-19) has disproportionately affected certain demographics in England, exacerbating existing health disparities. Effective therapeutics are a critical line of defence against COVID-19, particularly for patients at elevated risk for severe disease. Surveillance systems were established to monitor usage of COVID-19 therapeutics in hospital and community settings and inform stewardship. Three antiviral therapies: nirmatrelvir plus ritonavir (Paxlovid®), remdesivir (Veklury®), and molnupiravir (Lagevrio®); and two neutralising monoclonal antibody therapies (nMAbs): sotrovimab (Xevudy®) and casirivimab with imdevimab (Ronapreve®); were in use in England between July 2020 to April 2023. This paper aims to illuminate trends in the utilisation of COVID-19 therapeutics treatment in both hospital and community settings, stratified by the Index of Multiple Deprivation (IMD) in England. Chapter 3 of the English Surveillance Programme for Antimicrobial Utilisation and Resistance (ESPAUR) report 2022 to 2023 also discusses the epidemiological surveillance of these five directly-acting antiviral COVID-19 therapeutics use in England between 2022 to 2023.

Real world use of Tixagevimab/Cilgavimab pre-exposure prophylaxis of COVID-19 in immunocompromised individuals: data from the OCTOPUS study. (preprint)

We report real world use over time in immunocompromised subjects receiving tixagevimab/cilgavimab (T/C) pre-exposure prophylaxis (PrEP). Observational study on participants receiving T/C PrEP stratified: never had COVID-19 (NoC), hybrids (H) and breakthrough infections (BTIs) if COVID-19 before or after PrEP, respectively. Anti-RBD IgG and BA.5 neutralizing antibodies (nAbs), mucosal IgG, T-cell immunity at the administration of T/C (T0), 3 (T1), 6 (T2), and 9 (T3) months after, were measured. Comparison of markers in each group across timepoints, Poisson regression model for BTIs incidence rate ratios were performed. N=231 participants: median age 63 years (IQR 54.0-73.0), 84% hematological disease, median vaccine dose of three. N=72 NoC, 103 H and 56 (24%) BTIs, mostly mild/moderate, IR 4.2 (95%CI 3.2-5.4) BTIs/100 patients-months, no factors associated with. A significant increase of anti-RBD IgG at T1 was observed in all the groups, with a decline at T2. GMTs of anti-BA.5 nAbs were low at T1 for all the groups and around/below the cut off. No changes of IFN-γ. Overall, a mucosal response was observed at T1. An incidence of 24% of mild/moderate BTIs was observed. Anti-RBD IgG levels persistence was ensured, BA.5 nAbs were low/undetectable, cellular T immunity remained stable.

Wastewater Based Epidemiology for Viral Surveillance in an Endemic Perspective: Evidence and Challenges (preprint)

Wastewater-Based Epidemiology (WBE) is currently used to monitor not only the spread of the pandemic virus SARS-CoV-2 but also for other viruses in endemic conditions, particularly in the absence of syndromic surveillance. Continuous monitoring of sewage requires high costs and significant time investment, highlighting the need for standardized methods and structured monitoring strategies. In this context, we conducted weekly wastewater monitoring in northwestern Italy, targeting Human Adenovirus (HAdV), Norovirus Genogroup II (NoVggII), Enterovirus (EV), and SARS-CoV-2. Samples collected at the entrance of treatment plants were concentrated using PEG/NaCl precipitation and viral nucleic acids were extracted and detected by real-time (RT-)qPCR. NoVggII resulted the most identified target (84.4%), followed by HAdV, SARS-CoV-2, and EV. Only HAdV and EV exhibited seasonal peaks in spring and summer. SARS-CoV-2 results compared with data previously collected in the same study area (February 2021 to September 2021) revealed a shift from an epidemic to a pandemic status, likely due to the evolution of variants. In conclusion, WBE, using standardized methods and an efficient monitoring strategy, proves valuable for virus surveillance in pandemic and epidemic scenarios, enabling the identification of temporal-local distribution patterns that are useful for making informed public health decision.

A Model-based approach for the preliminary design of the SAR Upstream element for the Italian IRIDE EO constellation based on users’ demand (preprint)

Traditional approaches to the design of satellite constellations are typically related to technical conditions and financial effort. The use of optimization methods is limited to design specific technical performances of the single spacecraft, missing to resort to a holistic view of the whole mission including user needs. Given the growing significance of large Low Earth Orbit (LEO) constellations of small satellites in Earth Observation, there is a pressing need to develop design tools that can incorporate user needs into the overall preliminary design process of a constellation. This paper aims to formulate and preliminarily test a systematic model-based approach capable of integrating user needs within the entire preliminary design process of a constellation architecture. The purpose of the methodology is to identify cost-effective architectures for more detailed design studies and to propose optimal solutions to fulfill the variety of requests from users. It is composed of five major steps: formulation, enumeration, simulation, evaluation, and down-selection. As a case study, the proposed approach is implemented to define the preliminary architecture of the Synthetic Aperture Radar (SAR) sub-constellation within the IRIDE Italian program. Performance is assessed based on the percentage of satisfied user needs and the volume of downlinked data to ground stations. Five optimal SAR constellations are proposed. Results demonstrate that the approach provides a valuable tool for the optimal preliminary design of a satellite infrastructure, taking into account not only technical performance but also user needs throughout the entire design process.

Beyond for Better and for Worse: Expanding Environmental Sensitivity into a Post-Traumatic Growth perspective (preprint)

People differ in their responses to experiences with some showing a heightened Environmental Sensitivity (ES) for better and for worse. Highly sensitive people tend to get easily overwhelmed in adverse conditions but also to flourish in enriched environments. Yet, no studies have investigated whether people with a heightened ES may experience a positive outcome as well, in terms of Post-traumatic Growth (PTG), when a traumatic event occurs. This study provided a first empirical evidence regarding the relationship between ES and PTG on a general population of 2387 adults (age range: 18-88yy) surveyed online during the first Covid-19 lockdown. Correlations showed that ES was positively associated with PTG, though with a small effect size. Interaction effects from regression analyses provided evidence that the ES-PTG association was stronger when the individuals experienced anxiety to some extent, and not too much depression. To conclude, findings suggested highly sensitive people as not only more susceptible to adversities, but also more open to experience a growth when faced with challenging events. Identifying potential paths of growth in individuals who are more prone to negative feelings can have important implications for clinical practice as well as for theory by broadening our understanding of the concept of environmental sensitivity.

Utility of accessible SARS-CoV-2 specific immunoassays in vaccinated adults with a history of advanced HIV Infection (preprint)

Background. Accessible SARS-CoV-2-specific immunoassays may inform clinical management in people with HIV, particularly in case of persisting immunodysfunction. We prospectively studied their application in vaccine recipients with HIV, purposely including participants with a history of advanced HIV infection. Methods. Participants received one (n=250), two (n=249) or three (n=42) doses of the BNT162b2 vaccine. Adverse events were documented through questionnaires. Sample collection occurred pre-vaccination and a median of 4 weeks post-second dose and 14 weeks post-third dose. Anti-spike and anti-nucleocapsid antibodies were measured with the Roche Elecsys chemiluminescence immunoassays. Neutralising activity was evaluated using the GenScript cPAss surrogate virus neutralisation test, following validation against a Plaque Reduction Neutralization Test. T-cell reactivity was assessed with the Roche SARS-CoV-2 IFNγ release assay. Results. Primary vaccination (2 doses) was well tolerated and elicited measurable anti-spike antibodies in 202/206 (98.0%) participants. Anti-spike titres varied widely, influenced by previous SARS-CoV-2 exposure, ethnicity, intravenous drug use, CD4 counts and HIV viremia as independent predictors. A third vaccine dose significantly boosted anti-spike and neutralising responses, reducing variability. Anti-spike titres >15 U/mL correlated with neutralising activity in 136/144 paired samples (94.4%). Three participants with detectable anti-S antibodies did not develop neutralising responses post-third dose, yet displayed SARS-CoV-2 specific IFNγ responses. Conclusions: SARS-CoV-2 vaccination is well-tolerated and immunogenic in adults with HIV, with responses improving post-third dose. Anti-spike antibodies serve as a reliable indicator of neutralising activity. Discordances between anti-spike and neutralising responses may be accompanied by detectable IFN-γ responses, underlining the complexity of the immune response in this population.

Integrated environmental and clinical surveillance for the prevention of acute respiratory infections (ARIs) in indoor environments and vulnerable communities (Stell-ARI) (preprint)

Background The epidemiological relevance of viral acute respiratory infections (ARIs) has been dramatically highlighted by COVID-19. However, other viruses cannot be neglected, such as the influenza virus, respiratory syncytial virus, human adenovirus. These viruses thrive in closed spaces, influenced by human and environmental factors. High-risk closed communities are the most vulnerable settings, where the real extent of viral ARIs is often difficult to evaluate, due to the natural disease progression and case identification complexities. During the COVID-19 pandemic, wastewater-based epidemiology has demonstrated its great potential for monitoring the circulation and evolution of the virus in the environment. The “Prevention of ARIs in indoor environments and vulnerable communities” study (Stell-ARI) addresses the urgent need for integrated surveillance and early detection of ARIs within enclosed and vulnerable communities such as Long-Term Care Facilities (LTCFs), prisons and primary schools. The rapid transmission of ARIs in such environments underscores the importance of comprehensive surveillance strategies to minimise the risk of outbreaks and safeguard community health, enabling proactive prevention and control strategies to protect the health of vulnerable populations.Methods The Stell-ARI study consists of designing and validating tools for integrated clinical and environmental-based surveillance for each setting, coupled with analytical methods for environmental matrices. The study design encompasses the development of specialised clinical surveillance involving pseudonymized questionnaires and nasopharyngeal swabs for virus identification, while the environmental surveillance includes air and surface microbiological and chemical monitoring, and virological analysis of wastewater. Integrating this information and the collection of behavioural and environmental risk factors into predictive and risk assessment models will provide a useful tool for early warning, risk assessment and informed decision-making.Discussion This study seeks to integrate clinical, behavioural, and environmental data to establish and validate a predictive model and risk assessment tool for the early warning and risk management of viral ARIs in closed and vulnerable communities prior to the onset of an outbreak.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates (preprint)

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, theres a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Covid-19 related cognitive, structural and functional brain changes among Italian adolescents and young adults: a multimodal longitudinal case-control study (preprint)

Coronavirus disease 2019 (COVID-19) has been associated with brain functional, structural, and cognitive changes that persist months after infection. Most studies of the neurologic outcomes related to COVID-19 focus on severe infection and aging populations. Here, we investigated the neural activities underlying COVID-19 related outcomes in a case-control study of mildly infected youth enrolled in a longitudinal study in Lombardy, Italy, a global hotspot of COVID-19. All participants (13 cases, 27 controls, mean age 24 years) completed resting state functional (fMRI), structural MRI, cognitive assessments (CANTAB spatial working memory) at baseline (pre-COVID) and follow-up (post-COVID). Using graph theory eigenvector centrality (EC) and data-driven statistical methods, we examined differences in ECdelta (i.e., the difference in EC values pre- and post-COVID-19) and volumetricdelta (i.e., the difference in cortical volume of cortical and subcortical areas pre- and post-COVID) between COVID-19 cases and controls. We found that ECdeltasignificantly between COVID-19 and healthy participants in five brain regions; right intracalcarine cortex, right lingual gyrus, left hippocampus, left amygdala, left frontal orbital cortex. The left hippocampus showed a significant decrease in volumetricdelta between groups (p=0.041). The reduced ECdelta in the right amygdala associated with COVID-19 status mediated the association between COVID-19 and disrupted spatial working memory. Our results show persistent structural, functional and cognitive brain changes in key brain areas associated with olfaction and cognition. These results may guide treatment efforts to assess the longevity, reversibility and impact of the observed brain and cognitive changes following COVID-19.

COVID-19 vaccination acceptance and hesitancy in health and care workers and the general population: a systematic review and policy recommendations (preprint)

Introduction: The COVID-19 pandemic challenged the scientific community to find and develop a vaccine to fight the disease. However, and despite the vaccines developed thus far, problems with achieving high vaccine coverages have emerged, even among high-risk groups such as health and care workers (HCW). The lack of experience with such extensive vaccination created knowledge gaps in health policy and multisector decisions worldwide.  Objective: This systematic review aims to examine factors that influence HCW's adherence to COVID-19 vaccination and national policies to vaccinate the HCW and other target groups.  Methods: Searches were performed in the PubMed, Embase, Scopus, CINAHL, Web of Science, Lilacs, and WHO databases, besides Google Scholar for grey literature. The eligibility criterion for inclusion was being a member of the HCW. Vaccination was the target intervention, and the COVID-19 pandemic was the context. Qualitative synthesis used a meta-aggregation approach.  Results: Nineteen articles were included in the review, with study samples varying from 48 to 5,708 participants. Most of the evidence came from cross-sectional and qualitative studies. The main findings were related to vaccine hesitancy rather than acceptance. Factors associated with HCW vaccine hesitancy included subjective feelings such as safety concerns, related to rapid vaccine development, and insufficient testing. Non-adherence to vaccination by HCW can contribute to vaccine hesitancy in the general population. Countries have adopted few public policies to address this problem, and the main concern is whether to enforce vaccination and the extent to which measures are legal. Conclusion: Despite the large number of studies identified in the review, the quality of the evidence base remains weak. Skepticism, mistrust, and hesitancy toward vaccination are global issues that can jeopardize vaccination coverage. Governments need guidance to develop gender-specific policies to inform HCW and the public about the need to adhere to vaccination in the current and future pandemic scenarios.

Genome-wide Association Study of Long COVID (preprint)

Infections can lead to persistent or long-term symptoms and diseases such as shingles after varicella zoster, cancers after human papillomavirus, or rheumatic fever after streptococcal infections(1,2). Similarly, infection by SARS-CoV-2 can result in Long COVID, a condition characterized by symptoms of fatigue and pulmonary and cognitive dysfunction(3-5). The biological mechanisms that contribute to the development of Long COVID remain to be clarified. We leveraged the COVID-19 Host Genetics Initiative(6,7) to perform a genome-wide association study for Long COVID including up to 6,450 Long COVID cases and 1,093,995 population controls from 24 studies across 16 countries. We identified the first genome-wide significant association for Long COVID at the FOXP4 locus. FOXP4 has been previously associated with COVID-19 severity(6), lung function(8), and cancers(9), suggesting a broader role for lung function in the pathophysiology of Long COVID. While we identify COVID-19 severity as a causal risk factor for Long COVID, the impact of the genetic risk factor located in the FOXP4 locus could not be solely explained by its association to severe COVID-19. Our findings further support the role of pulmonary dysfunction and COVID-19 severity in the development of Long COVID.

The Role of VSL#3 in the Treatment of Fatigue and Other Symptoms in Long Covid-19 Syndrome: a Randomized, Double-blind, Placebo-controlled Pilot Study (DELong#3) (preprint)

Long COVID, also known as Post-acute COVID-19 Syndrome (PACS), is a chronic condition affecting individuals who have recovered from acute COVID-19. It is currently estimated that around 65 million people worldwide suffer from Long COVID. It is characterized by a range of symptoms, including fatigue, exertion intolerance, neurocognitive and sensory impairment, sleep disturbance, myalgia/arthralgia, and dysautonomia. Among them fatigue has emerged as a burdensome and pervasive issue, significantly impacting the quality of life and daily functioning of Long COVID patients. Alterations in the composition of the intestinal microbiota has been reported in COVID-19 patients. Dysbiosis persists even after several months of recovery from acute SARS-CoV-2 infection. Based on this evidence, we carried out a phase 3, randomized, double-blind, placebo-controlled trial aimed at evaluating the efficacy of VSL#3, a consortium of probiotic bacterial strains, in reducing fatigue and improving various aspects of patients' well-being in patients with Long COVID syndrome.

Multiple genetic control of anti-COVID-19 vaccine response by HLA locus (preprint)

Since the beginning of the anti-COVID-19 vaccination campaign, it has become evident that vaccinated subjects exhibit considerable inter-individual variability in the response to the vaccine that could be partly explained by host genetic factors. A recent study reported that the immune response elicited by the Oxford-AstraZeneca vaccine in individuals from the United Kingdom was influenced by a specific allele of the human leukocyte antigen gene HLA-DQB1. We performed a genome-wide association study to investigate the genetic determinants of the antibody response to the Pfizer-BioNTech vaccine in an Italian cohort of 1,351 subjects. We confirmed the involvement of the HLA locus and observed significant associations with variants in HLA-A gene. In particular, the HLA-A*03:01 was the most significantly associated with serum levels of anti-SARS-CoV-2 antibodies. These results support the hypothesis that HLA genes modulate the response to anti-COVID-19 vaccines and highlight the need for genetic studies in diverse populations.